U.S. patent application number 16/106451 was filed with the patent office on 2019-01-03 for magnetic nanoparticles for nucleic acid purification.
The applicant listed for this patent is Roche Molecular Systems, Inc.. Invention is credited to Jens Christian Bolle, Thomas Walter, Peter Wenzig.
Application Number | 20190002871 16/106451 |
Document ID | / |
Family ID | 50389781 |
Filed Date | 2019-01-03 |
United States Patent
Application |
20190002871 |
Kind Code |
A1 |
Bolle; Jens Christian ; et
al. |
January 3, 2019 |
MAGNETIC NANOPARTICLES FOR NUCLEIC ACID PURIFICATION
Abstract
The present invention relates to monodisperse silanized
ferrimagnetic iron oxide particles, a method for producing the same
and a method for independent generic binding of nucleic acid
molecules to the particles.
Inventors: |
Bolle; Jens Christian;
(Penzberg, DE) ; Walter; Thomas; (Penzberg,
DE) ; Wenzig; Peter; (Munich, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Molecular Systems, Inc. |
Pleasanton |
CA |
US |
|
|
Family ID: |
50389781 |
Appl. No.: |
16/106451 |
Filed: |
August 21, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14637014 |
Mar 3, 2015 |
10100301 |
|
|
16106451 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 20/28009 20130101;
B01J 20/103 20130101; C12N 15/1013 20130101; H01F 1/0054 20130101;
C12Q 1/6806 20130101; B01J 20/28016 20130101; B01J 20/06 20130101;
B01J 20/3293 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; B01J 20/28 20060101 B01J020/28; C12Q 1/6806 20180101
C12Q001/6806; B01J 20/32 20060101 B01J020/32; B01J 20/06 20060101
B01J020/06; B01J 20/10 20060101 B01J020/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2014 |
EP |
14157699.1 |
Claims
1. A method for producing a plurality of silanized ferrimagnetic
iron oxide particles for independent generic nucleic acid binding,
wherein the method comprises the steps of: (a) adding an iron(II)
salt to a liquid glycol to obtain a solution, (b) raising the pH of
the solution to a value of at least 9 such that a precipitate is
obtained, wherein during steps (a) and (b) a first temperature is
applied to the solution and wherein steps (a) and (b) the solution
is gassed with nitrogen, (c) mixing the solution comprising the
precipitate at a second temperature such that ferrimagnetic iron
oxide particles are obtained, and (d) contacting the ferrimagnetic
iron oxide particles with a silicate solution such that silanized
ferrimagnetic iron oxide particles are obtained.
2. The method of claim 1, wherein contacting the ferrimagnetic iron
oxide particles with the silicate solution comprises the steps of:
(d1) sonificating the silicate solution comprising the
ferrimagnetic iron oxide particles, (d2) lowering the pH of the
silicate solution to a value of 6 or below such that silanized
ferrimagnetic iron oxide particles are obtained, (d3) washing of
the silanized ferrimagnetic particles with water, and (d4) washing
of the silanized ferrimagnetic particles with isopropanol such that
crosslinking occurs within the silicate layer.
3. The method of claim 1, wherein the iron(II) salt is soluble in
the liquid glycol and wherein the iron(II) salt is selected from
the group consisting of FeCl.sub.2, FeSO.sub.4, FeAc.sub.2 and the
hydrated forms thereof.
4. The method of claim 1, wherein the liquid glycol is triethylene
glycol.
5. The method of claim 1, wherein the pH of the solution in step
(b) is raised to a value of 10.5 using sodium hydroxide.
6. The method of claim 1, wherein the first temperature has a value
from 20 to 120.degree. C. and wherein the first temperature is
maintained for 5 to 60 min.
7. The method of claim 1, wherein the second temperature has a
value from 150 to 300.degree. C. and wherein the second temperature
is maintained for 20 min to 48 h.
8. The method of claim 1, wherein the silicate is selected from the
group consisting of potassium silicate and sodium silicate and
wherein the silicate is present in the solution in a concentration
from 1 to 30% w/v.
9. The method of claim 2, wherein the pH of the solution in step
(d2) is lowered to a value of 5 using hydrochloric acid.
10. The method of claim 1, wherein step (d) is repeated one or more
times.
11. A composition comprising monodisperse silanized ferrimagnetic
iron oxide particles for independent generic nucleic acid binding
wherein said particles comprise: (a) a core wherein the core
comprises an inner layer comprising Fe.sub.3O.sub.4 and an outer
layer comprising Fe.sub.2O.sub.3, (b) a coating wherein the coating
comprises silica and silicates from sodium silicate precipitation,
wherein said particles display a sedimentation speed in pure water
of less than 60 .mu.m/s, and no significant iron bleeding in 1M HCl
for at least 60 minutes.
12. The composition of claim, wherein the difference in size of the
monodisperse silanized ferrimagnetic iron oxide particles is in
average smaller than 5%.
13. The composition of claim 11, wherein the size of the particles
is between 20 nm and 600 nm.
14. A method for independent generic binding of nucleic acid
molecules to a composition comprising: contacting a sample
containing nucleic acid molecule to the composition, wherein the
composition comprises monodisperse silanized ferrimagnetic iron
oxide particles, wherein said particles comprise a core, wherein
the core comprises an inner layer comprising Fe.sub.3O.sub.4, and
an outer layer comprising Fe.sub.2O.sub.3, a coating, wherein the
coating comprises silica and silicates from sodium silicate
precipitation, wherein the particles display a sedimentation speed
in pure water of less than 60 .mu.m/s, and no significant iron
bleeding in 1M HCl for at least 60 minutes.
15. The method of claim 14, wherein the nucleic acid molecules are
DNA molecules or RNA molecules.
16. The composition of claim 11, wherein the nucleic acid is a
nucleic acid molecule that a DNA molecule and/or RNA molecule.
Description
CROSS REFERENCE TO RELATED INVENTION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119 of EP 14157699.1, filed Mar. 4, 2014, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to monodisperse silanized
ferrimagnetic iron oxide particles, a method for producing the same
and their use for independent generic binding nucleic acid
molecules.
BACKGROUND OF THE INVENTION
[0003] Magnetic nanoparticles are widly used in the field of
nucleic acid purification. All commercially available large scale
magnetic nanoparticles have superparamagnetic properties. In
contrast, ferrimagnetic nanoparticles are not commercially
available and only known from academic publications. Such
publications include nanoparticles with silica coatings (Chen et
al; J. of alloys and compounds 497 (2010) 221-227; Wang et al;
Bioresource Technology 101 (2010) 8931-8935; Reza et al; Cent. Eu.
J. Chem 5 (2010) 1041-1048). One of the major drawbacks of the
majority of preparations of ferrimagnetic nanoparticles known in
the art where low pressure reactors at <100.degree. C. are used
is that the synthesis is difficult to upscale and automate (Wang et
al; Bioresource Technology 101 (2010) 8931-8935; Reza et al; Cent.
Eu. J. Chem 5 (2010) 1041-1048).
[0004] The use of glycols as solvent and reducing agent for the
synthesis of ferrimagnetic nanoparticles has been shown (Wiley et
al; Nano Lett. 4 (2004) 1733-1739; D. Larcher, R. J. Partrice; J.
Solid State Chem. 154 (2000) 405-411; Gai et al; J. Phys. D: Appl.
Phys. 43 (2010) 445-553)--also in combination with the
surfactant-free synthesis route ("Green synthesis route") (Liu et
al; Eur. J. Inorg. Chem. 2 (2010) 4499-4505). These preparations
are focused on the use of iron(III) salts which result in poor size
distribution and yield when upscaled to more than 100 mL scale.
[0005] Silanization of ferrimagnetic nanoparticles is only known in
the art according to the Stober method which relies on harmful
alkoxy silanes. Using harmless SiO.sub.2 solution in purely aqueous
conditions have only been shown in the context of superparamagnetic
particles (Philipse et al; Langmuir 10 (1994) 92-99).
[0006] Extraction of nucleic acids by means of hydrothermally
prepared ferrimagnetic particle structures are described (Gai et
al; J. Phys. D: Appl. Phys. 43 (2010) 445-553). These publications
used ferrimagnetic particles which were produced by silane
chemistry or without silicone containing coating, wherein the
production was complex or resulted in a poor eluation.
[0007] The object of the present invention is the provision of
monodisperse silanized ferrimagnetic iron oxide particles for
nucleic acid binding which do not show the above mentioned
drawbacks and a method for producing the same.
SUMMARY OF THE INVENTION
[0008] It was found that silanized ferrimagnetic iron oxide
particles for independent generic nucleic acid binding can be
produced with a high degree of uniformity (very defined
reproducible diameters of the particles), a high yield and
homogeniously high magnetization saturations. Furthermore it was
found that silanizing can be performed resulting in a very dense
layer of silicate around the magnetite core of the particles, such
that the quality of silanization is compareable to the quality
reached by known processes, however, with less harmful chemicals
and more cost effective educts as compared to the known
processes.
[0009] The present invention thus relates to a method for producing
a plurality of silanized ferrimagnetic iron oxide particles for
independent generic nucleic acid binding, wherein the method
comprises the steps of a) adding an iron(II) salt to a liquid
glycol to obtain a solution, b)raising the pH of the solution to a
value of at least 9 such that a precipitate is obtained, wherein
during steps a) and b) a first temperature is applied to the
solution and wherein during steps a) and b) the solution is gassed
with nitrogen, c) mixing the solution comprising the precipitate at
a second temperature such that ferrimagnetic iron oxide particles
are obtained, and d) contacting the ferrimagnetic iron oxide
particles with a silicate solution such that silanized
ferrimagnetic iron oxide particles are obtained. In one embodiment
the step of contacting the ferrimagnetic iron oxide particles with
a silicate solution comprises the steps of d1) sonificating the
silicate solution comprising the ferrimagnetic iron oxide
particles, d2) lowering the pH of the silicate solution to a value
of 6 or below such that silanized ferrimagnetic iron oxide
particles are obtained, d3) washing of the silanized ferrimagnetic
particles with water, and d4) washing of the silanized
ferrimagnetic particles with isopropanol such that crosslinking
occurs within the silicate layer.
[0010] The present invention further relates to monodisperse
silanized ferrimagnetic iron oxide particles for independent
generic nucleic acid binding comprising a core, wherein the core
comprises an inner layer comprising Fe.sub.3O.sub.4 and an outer
layer comprising Fe.sub.2O.sub.3, a coating, wherein the coating
comprises silica and silicates from sodium silicate precipitation,
wherein the particles display a sedimentation speed in pure water
of less than 60 .mu.m/s, and no significant iron bleeding particles
in 1M HCl for at least 60 min.
[0011] The present invention further relates to a method for
independent generic binding of nucleic acid molecules to a
composition comprising, contacting a sample containing nucleic acid
molecule to the composition, wherein the composition comprises
monidisperse silanized ferrimagnetic iron oxice particles, wherein
said particles comprise a core, wherein the core comprises an inner
layer comprising Fe.sub.3O.sub.4 and an outer layer comprising
Fe.sub.2O.sub.3, a coating, wherein the coating comprises silica
and silicates from sodium silicate precipitation, wherein the
particles display a sedimentation speed in pure water of less than
60 .mu.m/s, and no significant iron bleeding in 1M HCl for at least
60 minutes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 Shows the regulation temperature and the resulting
temperature of the reactor.
[0013] FIG. 2 Shows the labeled parts of the disassembled
stirrer.
[0014] FIG. 3 Shows the manometer, temperature sensor,
N.sub.2-pipe, needle valve and burst protection and corresponding
parts.
[0015] FIG. 4 Shows the core of the top panel and corresponding
parts.
[0016] FIG. 5 Schematic drawing of the special design glas reactor
used for silanizing the ferrimagnetic iron oxide particles.
(Ar=Argon, M=Motor, US=Ultrasonic probe).
DETAILED DESCRIPTION OF THE INVENTION
[0017] The following definitions are set forth to illustrate and
define the meaning and scope of various terms used to describe the
invention herein.
[0018] The terms "a", "an" and "the" generally include plural
referents, unless the context clearly indicates otherwise.
[0019] The term "acid" is used herein as known to the expert
skilled in the art and refers to a substance capable of donating a
proton in polar or non-polar solvents. The acid of choice for a
particular reaction depends on the starting materials, the solvent
and the temperature used for a specific reaction. Examples of acids
include phosphoric acid, sulphuric acid and hydrochloric acid.
[0020] The term "alkyl" denotes a monovalent linear or branched
saturated hydrocarbon group of 1 to 12 carbon atoms. In particular
embodiments, alkyl has 1 to 7 carbon atoms, and in more particular
embodiments 1 to 4 carbon atoms. Examples of alkyl include methyl,
ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, or
tert-butyl.
[0021] In the context of the pH the below terms are defined as
follows: [0022] "At least": A "pH of at least 7" refers to a pH
value of 7 or higher, e.g. a pH value of 7, 8, 9, 10, 11, 12, 13 or
14. [0023] "Raise": "Raising the pH" refers to changing the pH from
a lower value to a higher value, e.g. changing the pH from 7 to 8.
[0024] "Or below": A "pH of 7 or below" refers to a pH value of 7
or lower, e.g. a pH value of 1, 2, 3, 4, 5, 6 or 7. [0025] "Lower":
"Lowering the pH" refers to changing the pH from a higher value to
a lower value, e.g. changing the pH from 8 to 7.
[0026] The term "base" is used herein as known to the expert
skilled in the art and refers to a substance capable of accepting a
proton in polar or non-polar solvents. The base of choice for a
particular reaction depends on the starting materials, the solvent
and the temperature used for a specific reaction. Examples of bases
include carbonate salts such as potassium carbonate, potassium
bicarbonate, sodium carbonate, sodium bicarbonate, and cesium
carbonate; halides such as cesium fluoride; phosphates such as
potassium phosphate, potassium dihydrogen phosphate, and potassium
hydrogen phosphate; hydroxides such as lithium hydroxide, sodium
hydroxide, and potassium hydroxide; disilylamides such as lithium
hexamethyldisilazide, potassium hexamethyldisilazide, and sodium
hexamethyldisilazide; trialkylamines such as triethylamine,
diisopropylamine, and diisopropylethylamine; heterocyclic amines
such as imidazole, pyridine, pyridazine, pyrimidine, and pyrazine;
bicyclic amines such as DBN and DBU; and hydrides such as lithium
hydride, sodium hydride, and potassium hydride. Examples of bases
include alkali metal hydroxides as defined herein; alkali metal
hydrides such as lithium, sodium, or potassium hydride; and
nitrogen-containing bases such as lithium diisopropyl amide (LDA),
lithium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide,
and potassium bis(trimethylsilyl)amide; and the like. It will be
apparent to a skilled practioner that individual base and solvent
combinations can be preferred for specific reaction conditions
depending on such factors as the solubility of reagents, reactivity
of reagents with Isomidazolam or the solvent, and preferred
temperature ranges.
[0027] The term "crosslinking" as used herein refers to a chemical
process, wherein two or more molecules interact to form n-mers,
wherein n>1. Such interaction can be a covalent bond,
hydrophilic or hydrophobic, ionic or electrostatic,
interaction.
[0028] The term "ferrimagnetic" as used herein refers to a material
consisting of populations of atoms with opposing but unequally
distributed magnetic moments. Thus resulting in a magnetic
saturation and remanence once an external magnetic field was
applied.
[0029] The term "independent generic nucleic acid binding" as used
herein refers to binding of single stranded and/or double stranded
nucleic acid molecules such as DNA and/or RNA. Binding of such
molecules occur independently of properties of the nucleic acid
molecules, such sequence, presence or absence of modifications and
concentration.
[0030] The term "iron bleeding" as used herein refers to the
solvation of iron ions into the surrounding solvation medium. The
term "significant iron bleeding" as used herein refers to a iron
ion concentration in the surrounding solvent detectable by light
spectroscopic methods (UV/Vis-Spectroscopy). Spectroscopy was
performed from 200 nm to 800 nm in a quarz glas cuvette with 1 cm
transmission distance in a total volume of 3 mL 5% w/w particle
after adding 5M HCl solution and shaking for 10 seconds followed by
magnetic separation of the beads by using a Neodymium-Iron-Borum
magnet (1 cm.times.1 cm.times.1 cm). After the blanking with pure
water no peak larger than the background noise should be seen or
identified by the analysis software over the whole spectrum.
[0031] The term "monodisperse" as used. herein refers to particles
of essentially the same size. The size of the monodisperse
silanized ferrimagnetic iron oxide particles of one certain batch
is essentially the same for all particles within that batch.
Essentially the same size of the particles has to be interpreted in
the context of this description such that the difference in size,
i.e. difference in diameter, of the particles is in average smaller
than 5% (coefficient of variation).
[0032] The terms "nucleic acid", "nucleic acid molecule" or
"polynucleotide" can be used interchangeably and refer to a polymer
that can be corresponded to a ribose nucleic acid (RNA) or
deoxyribose nucleic acid (DNA) polymer, or an analog thereof. This
includes polymers of nucleotides such as RNA and DNA, as well as
synthetic forms, modified (e.g., chemically or biochemically
modified) forms thereof, and mixed polymers (e.g., including both
RNA and DNA subunits). Exemplary modifications include methylation,
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as uncharged
linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, carbamates, and the like), pendent moieties (e.g.,
polypeptides), intercalators (e.g., acridine, psoralen, and the
like), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids and the like). Also included are synthetic
molecules that mimic polynucleotides in their ability to bind to a
designated sequence via hydrogen bonding and other chemical
interactions. Typically, the nucleotide monomers are linked via
phosphodiester bonds, although synthetic forms of nucleic acids can
comprise other linkages (e.g., peptide nucleic acids as described
in Nielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can
be or can include, e.g., a chromosome or chromosomal segment, a
vector (e.g., an expression vector), an expression cassette, a
naked DNA or RNA polymer, the product of a polymerase chain
reaction (PCR), an oligonucleotide, a probe, and a primer. A
nucleic acid can be, e.g., single-stranded, double-stranded, or
triple-stranded and is not limited to any particular length. Unless
otherwise indicated, a particular nucleic acid sequence comprises
or encodes complementary sequences, in addition to any sequence
explicitly indicated.
[0033] The term "plurality" as used herein refers to two or more
items or components. Accordingly, the term "plurality of particles"
refers to two or more particles, such as silanized ferromagnetic
iron oxide particles.
[0034] As used herein, the term "precipitate" refers to the for
ration of solid, such as a particle, in a solution during a
chemical reaction.
[0035] The term "sedimentation speed" as used herein refers to the
speed (length/time) with which a particle, such as a silanized
ferromagnetic iron oxide particle, sediments at a defined
gravitational force within a liquid, such as pure water. If no
value for a defined gravitational force is given, the gravitational
force to be assumed for the given reaction is gravitation of earth,
i.e. 1.0 g.
[0036] The term "silanized" as used herein refers to the formation
of a top layer containing silicone which is crosslinked by
oxygen.
[0037] In one aspect, the description refers to a method for
producing a plurality of silanized ferrimagnetic iron oxide
particles for independent generic nucleic acid binding, wherein the
method comprises the steps of a) adding an iron(II) salt to a
liquid glycol to obtain a solution, b) raising the pH of the
solution to a value of at least 9 such that a precipitate is
obtained, wherein during steps a) and b) a first temperature is
applied to the solution and wherein during steps a) and b) the
solution is gassed with nitrogen, c) mixing the solution comprising
the precipitate at a second temperature such that ferrimagnetic
iron oxide particles are obtained, and d) contacting the
ferrimagnetic iron oxide particles with a silicate solution such
that silanized ferrimagnetic iron oxide particles are obtained.
[0038] The addition of the iron(II) salt to the liquid glycol to
obtain a solution as performed in step a) above has the advantage
over the methods known in the art that it results in a better size
control. Furthermore, gassing the solution with nitrogen leads to a
higher yield.
[0039] In one embodiment, the step of contacting the ferrimagnetic
iron oxide particles with a silicate solution comprises the steps
of d1) sonificating the silicate solution comprising the
ferrimagnetic iron oxide particles, d2) lowering the pH of the
silicate solution to a value of 6 or below such that silanized
ferrimagnetic iron oxide particles are obtained, d3) washing of the
silanized ferrimagnetic particles with water, and d4) washing of
the silanized ferrimagnetic particles with isopropanol such that
crosslinking occurs within the silicate layer.
[0040] The crosslinking performed in step d4) results in a very
dense layer of silicate around the magnetite core and in a
silanization compareable to the Stober method. However, the
crosslinking according to the present description is performed with
harmless chemicals and cost effective educts.
[0041] In one embodiment, the iron(II) salt is soluble in the
liquid glycol. In a specific embodiment, the iron(II) salt soluble
in the liquid glycol is selected from the group consisting of
FeCl.sub.2, FeSO.sub.4, FeAc.sub.2 and the hydrated forms thereof.
The term hydrated forms as used herein has to be understood as a
compound which is formed by the addition of water. Possible
hydrated forms according to the present description are
FeCl.sub.2.n H.sub.2O, FeO.sub.4.n H.sub.2O, FeAc.sub.2.n H.sub.2O,
wherein n.gtoreq.1. Specific hydrated forms according to the
present description are FeCl.sub.2.4 H.sub.2O, FeSO.sub.4.4
H.sub.2O, FeAc.sub.2.4 H.sub.2O. In a specific embodiment, the
iron(II) salt soluble in the liquid glycol is FeCl.sub.2 and
hydrated forms thereof.
[0042] In one embodiment, the concentration of FeCl.sub.2.4
H.sub.2O in the liquid glycol is from 50 mmol to 70 mmol. In a
specific embodiment, the concentration of FeCl.sub.2.4 H.sub.2O in
the liquid glycol is from 55 mmol to 65 mmol. In a more specific
embodiment, the concentration of FeCl.sub.2.4 H.sub.2O in the
liquid glycol is from 58 mmol to 62 mmol. In a specific embodiment,
the concentration of FeCl.sub.2.4 H.sub.2O in the liquid glycol is
61.9 mmol.
[0043] In a specific embodiment, the liquid glycol is an alkyl
glycol and polymerized forms thereof. In a specific embodiment, the
alkyl glycol is selected from the group consisting of ethylene
glycol, diethylene glycol, triethylene glycol, tetraethylene
glycol, propylene glycol, dipropylene glycol, tripropylene glycol,
tetraproylene glycol, butylene glycol, dibutylene glycol,
tributylene glycol, tetrabutylene glycol. In a more specific
embodiment, the alkyl glycol is triethylene glycol.
[0044] In one embodiment, the pH of the solution in step b) is
raised to a value of at least 9. In a specific embodiment, the pH
of the solution in step b) is raised to a value of at least 10. In
a more specific embodiment, the pH of the solution in step b) is
raised to a value of 10.5.
[0045] In one embodiment, the pH of the solution in step b) is
raised using a base. In a specific embodiment, the pH of the
solution in step b) is raised using an alkaline metal hydroxide. In
a more specific embodiment, the pH of the solution in step b) is
raised using an alkaline metal hydroxide selected from the group
consisting of lithium hydroxide, sodium hydroxide and potassium
hydroxide.
[0046] In one embodiment, the first temperature has a value from 20
to 120.degree. C. In a specific embodiment, the first temperature
has a value from 40 to 120.degree. C., from 60 to 120.degree. C.,
from 80 to 120.degree. C., or from 90 to 110.degree. C. In a more
specific embodiment, the first temperature has a value from 95 to
105.degree. C. In an even more specific embodiment, the first
temperature has a value of 100.degree. C. The application of the
first temperature results in the evaporation of excess water from
the reaction and thus in higher magnetization rates due to less
uncontrolled further oxidation of the magnetite produced during the
reaction.
[0047] In one embodiment, the first temperature is maintained for 5
to 60 min. In specific embodiment, the first temperature is
maintained for 10 to 50 min. In a more specific embodiment, the
first temperature is maintained for 20 to 50 min, for 30 to 50 min
or for 35 to 45 min. In an even more specific embodiment, the first
temperature is maintained for 40 min.
[0048] In one embodiment the gassing with nitrogen is performed at
a pressure of between 0.1 and 1.0 bar at the outlet valve. In a
specific embodiment, the gassing with nitrogen is performed at a
pressure of between 0.2 and 0.8 bar at the outlet valve. In a more
specific embodiment, the gassing with nitrogen is performed at a
pressure of between 0.3 and 0.6 bar at the outlet valve. In an even
more specific embodiment, the gassing with nitrogen is performed at
a pressure of between 0.35 and 0.45 bar at the outlet valve. In a
specific embodiment, the gassing with nitrogen is performed at a
pressure of 0.4 bar at the outlet valve. Gassing with nitrogen at
the above mentioned pressure values reduces the oxygen present in
the solution and thus reduces undesirable oxidation during
precipitation of the particles. Thus, higher yields and
homogeniously high magnetization saturations are achieved.
[0049] In one embodiment, the second temperature has a value from
150 to 300.degree. C. In a specific embodiment, the second
temperature has a value from 200 to 300.degree. C., from 210 to
290.degree. C., from 220 to 280.degree. C., from 230 to 270.degree.
C. or from 240 to 260.degree. C. In a more specific embodiment, the
second temperature has a value of 250.degree. C. The second
temperature results in particles with defined reproducible
diameters even at scales larger than 500 mL.
[0050] In one embodiment, the second temperature is maintained for
20 min to 48 h. In a specific embodiment, the second temperature is
maintained for 20 min to 40 h, for 20 min to 30 h, for 20 min to 20
h, for 20 min to 10 h, for 20 min to 5 h, for 20 min to 2 h, for 30
min to 90 min, for 40 min to 90 min, for 50 min to 90 min, for 60
min to 90 min or for 70 min to 90 min. In a more specific
embodiment, the second temperature is maintained for 80 min.
[0051] In a specific embodiment, the second temperature is
regulated according to the following protocol. The second
temperature starts at a value of 80.degree. C. and is increased to
a value of 250.degree. C. within a time period of 20 min. The value
of 250.degree. C. is maintained for a time period of 80 min.
Subsequently, the second temperature is decreased to a value of
30.degree. C. within a time period of 35 min. The second
temperature refers to the temperature within the reactor. The
temperature gradient within the reactor and the actual regulation
temperature is depicted in FIG. 1. The gradient as shown in the
figure has to be accurately executes such that the controlled
environment ensures the control of the reaction for forming
particles with a homogeneous size distribution and high
magnetization saturations.
[0052] In one embodiment, the silicate is selected from the group
consisting of potassium silicate and sodium silicate. In a specific
embodiment, the silicate is sodium silicate.
[0053] In one embodiment, the silicate is present in the solution
in a concentration from 1 to 30% w/v. In a specific embodiment, the
silicate is present in the solution in a concentration from 1 to
20% w/v, from 1 to 15% w/v, from 1 to 15% w/v, from 1 to 10% w/v,
from 3 to 7% w/v or from 4 to 6% w/v. In a specific embodiment, the
silicate is present in the solution in a concentration of 5.3%
w/v.
[0054] In one embodiment, sonificating is performed with an
amplitude of 60 to 100%. In a specific embodiment, sonificating is
performed with an amplitude of 70 to 90%. In a more specific
embodiment, sonificating is performed with an amplitude of 75 to
85%. In an even more specific embodiment, sonificating is performed
with an amplitude of 80%. In one embodiment, sonificating is
performed with a cycle of 10 to 30%. In a specific embodiment,
sonificating is performed with a cycle of 15 to 25%. In a more
specific embodiment, sonificating is performed with a cycle of 18
to 22%. In an even more specific embodiment, sonificating is
performed with a cycle of 20%. In a specific embodiment,
sonificating is performed with 80% of amplitude and 20% of
cycle.
[0055] In one embodiment, sonificating is performed for a duration
of 1 to 10 seconds. In a specific embodiment, sonificating is
performed for a duration of 3 to 8 seconds. In a more specific
embodiment, sonificating is performed for a duration of 4 to 6
seconds. In an even more specific embodiment, sonificating is
performed for a duration of 5 seconds. In one embodiment, the
sonificating pulses are interrupted by a pause of 30 to 70 seconds.
In a specific embodiment, the sonificating pulses are interrupted
by a pause of 40 to 60 seconds. In a more specific embodiment, the
sonificating pulses are interrupted by a pause of 45 to 55 seconds.
In an even more specific embodiment, the sonificating pulses are
interrupted by a pause of 50 seconds. In a specific embodiment,
sonificating is performed for a duration of 5 seconds followed by a
pause of 50 seconds.
[0056] In one embodiment, the pH of the solution in step d2) is
lowered to a value of 7 or below. In a specific embodiment, the pH
of the solution in step d2) is lowered to a value of 6 or below. In
a more specific embodiment, the pH of the solution in step d2) is
lowered to a value of 5.
[0057] In one embodiment, the pH of the solution in step d2) is
lowered using an acid selected from the group consisting of
phosphoric acid, sulphuric acid and hydrochloric acid. In a
specific embodiment, the pH of the solution in step d2) is lowered
using any non-oxidizing and water-soluble acid. In a specific
embodiment, the pH of the solution in step d2) is lowered using a
non-oxidizing and water-soluble acid selected from the group
consisting of hydrochloric acid, boric acid or prussic acid. In a
specific embodiment, the pH of the solution in step d2) is lowered
using hydrochloric acid. In another specific embodiment, the pH of
the solution in step d2) is lowered using 1M hydrochloric acid.
[0058] In another embodiment, the method for producing a plurality
of silanized ferrimagnetic iron oxide particles for independent
generic nucleic acid binding comprises the steps of a) adding
FeCl.sub.2 or the hydrated forms thereof to triethylene glycol to
obtain a solution, b) raising the pH of the solution to a value of
10.5 with sodium hydroxide such that a precipitate is obtained,
wherein during steps a) and b) a first temperature is applied to
the solution, wherein the first temperature has a value of
100.degree. C. and is maintained for 40 min, and wherein during
steps a) and b) the solution is gassed with nitrogen at a pressure
of 0.4 bar, c) mixing the solution comprising the precipitate at a
second temperature, wherein the second temperature has a value of
250.degree. C. and is maintained for 80 min, such that
ferrimagnetic iron oxide particles are obtained, and d) contacting
the ferrimagnetic iron oxide particles with a solution of 5.3% w/v
sodium silicate such that silanized ferrimagnetic iron oxide
particles are obtained, wherein the step of contacting the
ferrimagnetic iron oxide particles with the silicate solution
comprises the steps of d1) sonificating the silicate solution
comprising the ferrimagnetic iron oxide particles, d2) lowering the
pH of the silicate solution to a value of 5 such that silanized
ferrimagnetic iron oxide particles are obtained, d3) washing of the
silanized ferrimagnetic particles with water, and d4) washing of
the silanized ferrimagnetic particles with isopropanol such that
crosslinking occurs within the silicate layer.
[0059] In some embodiments of the method described herein, step d)
is repeated one or more times. Between the respective repetitions,
the ferrimagnetic iron oxide particles are washed with a wash
solution. Hence, in some embodiments, the method described herein
comprises after step d) the additional step e): washing the
silanized ferrimagnetic iron oxide particles one or more times with
a wash solution. In some embodiments, the wash buffer in step e) is
water, in some embodiments distilled or deionized water. In further
embodiments, the wash buffer is isopropanol or an equivalent
alcohol. The sequence of steps d) and e) is in some embodiments
repeated one or more times. Where step d) comprises steps d1) to
d4), the above-mentioned embodiments apply to the respective
sequence of substeps from d1) to d4).
[0060] The repetition of step d) or the sequence of steps d) and
e), respectively, result in particles with multiple layers of
silica and silicates from sodium silicate precipitation. As shown
in Example 4, such multiple-layered silanized ferrimagnetic iron
oxide particles display enhanced nucleic acid binding
properties.
[0061] In another aspect, the present invention relates to
monodisperse silanized ferrimagnetic iron oxide particles for
independent generic nucleic acid binding having the following
characteristics: A core, wherein the core comprises an inner layer
comprising Fe.sub.3O.sub.4 and an outer layer comprising
Fe.sub.2O.sub.3, a coating, wherein the coating comprises silica
and silicates from sodium silicate precipitation, a sedimentation
speed in pure water of less than 60 .mu.m/s, and no significant
iron bleeding occurs on the particles in 1M HCl for at least 60
min.
[0062] In one embodiment, the difference in size of monodisperse
silanized ferrimagnetic iron oxide particles is in average smaller
than 5%. In a specific embodiment, the size of the particles is
between 20 nm to 600 nm. In another embodiment, the size of the
particles is 100 nm. In still another embodiment, the diameter of
the particles is 100 nm. The size of the particles of one certain
batch can be varied by adjusting the concentration of iron(II) salt
in the liquid glycol. This however has to be understood that the
size of the silanized ferrimagnetic iron oxide particles of one
certain batch is essentially the same for all particles within that
batch. Essentially the same size of the particles has to be
interpreted in the context of this description such that the
difference in size of the particles is in average smaller than
5%.
[0063] The monodisperse silanized ferrimagnetic iron oxide
particles according to the description appear black in suspension.
In one embodiment, the sedimentation speed of the monodisperse
silanized ferrimagnetic iron oxide particles in pure water is less
than 100 .mu.m/s, less than 90 .mu.m/s, less than 80 .mu.m/s, less
than 70 .mu.m/s, less than 60 .mu.m/s or less or equal than 50
.mu.m/s. In a specific embodiment, the sedimentation speed of the
monodisperse silanized ferrimagnetic iron oxide particles in pure
water is 50 .mu.m/s.
[0064] In one embodiment, the yield of the synthesis of the
monodisperse silanized ferrimagnetic iron oxide particles is at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%
or at least 70%. In a specific embodiment, the yield of the
synthesis of the monodisperse silanized ferrimagnetic iron oxide
particles is at least 75%.
[0065] As an alternative or an addition, for monodisperse silanized
ferrimagnetic iron oxide particles that have multiple layers of
coating as described in the context of the production method, the
relative amount of coating on a specific particle or population of
particles may be varied. For instance, the mass of coating may be,
for instance, 20%, 40%, 60%, 80%, 100%, 120%. 140%, 160%, 180%,
200%, or other percentages relative to the mass of the respective
iron oxide core. Such particles are used in Example 4.
[0066] In another aspect, the present invention relates to the use
of the monodisperse silanized ferrimagnetic iron oxide particles as
described in the previous paragraph for independent generic binding
nucleic acid molecules. In one embodiment, the nucleic acid
molecules are selected from the group consisting of RNA molecules
and DNA molecules. In a specific embodiment, the nucleic acid
molecules are DNA molecules. In a further embodiment, the nucleic
acid molecules are RNA molecules.
[0067] As decribed supra, the binding of nucleic acids to magnetic
particles is often part of a nucleic acid isolation or extraction
procedure from essentially any source, such as cultured
microorganisms, uncultured microorganisms, complex biological
mixtures, tissues, sera, ancient or preserved tissues,
environmental isolates or the like or from any "sample" that
contains nucleic acid. Typically, one of the first steps of
purification of a biological target material comprises releasing
the contents of cells or viral particles by using enzymes and/or
chemical reagents. This process is commonly referred to as "lysis".
The nucleic acid to be isolated is ideally essentially unaffected
by the lysis step. As known in the art, lysis procedures can
involve chaotropic agents, ionic and/or non-ionic detergents such
as SDS or sarcosyl, mechanic disruption by shearing forces or the
like, French Press, ultrasound, liquid nitrogen, enzymes such as
lysozyme or proteases, freeze-drying, heat or osmotic shock, cell
membrane disruption under alkaline conditions, or other measures
known by the person skilled in the art.
[0068] The released nucleic acid may then be bound to suitable
binding particles such as the silanized ferrimagnetic iron oxide
particles described herein. This binding step may involve the
presence of chaotropic agents.
[0069] "Chaotropic agents" are substances that generally disturb
the ordered structure of water molecules in solution and
non-covalent binding forces in and between molecules. They can make
several contributions to the isolation procedure. Chaotropic agents
also contribute to the disruption of biological membranes, such as
plasma membranes or the membranes of cell organelles, if present.
Useful chaotropic agents in the context of the present invention
include, but are not limted to, guanidinium salts such as
guanidinium thiocyanate, guanidinium hydrochloride, guanidinium
chloride or guanidinium isothiocyanate, urea, perchlorates such as
potassium perchlorate, other thiocyanates or potassium iodide or
sodium iodide. They can be applied as RNase inhibitors by
disturbing the nuclease's tertiary structure. Usually, no further
RNase inhibitor need to be applied to the lysis buffer when the
biological target material is a nucleic acid. Also, chaotropic
agents can play a significant role in the adhesive binding of
nucleic acids to surfaces like glass. For lysis and/or binding
purposes, chaotropic agents can be applied at a concentration of
about 2 to about 8 M, and in some embodiments at a concentraiton of
about 4 to about 6 M.
[0070] Further embodiments are included by the following items:
[0071] 1. Method for producing a plurality of silanized
ferrimagnetic iron oxide particles for independent generic nucleic
acid binding, wherein the method comprises the steps of: [0072] a.
Adding an iron(II) salt to a liquid glycol to obtain a solution,
[0073] b. Raising the pH of the solution to a value of at least 9
such that a precipitate is obtained, [0074] wherein during steps a)
and b) a first temperature is applied to the solution and wherein
during steps a) and b) the solution is gassed with nitrogen, [0075]
c. Mixing the solution comprising the precipitate at a second
temperature such that ferrimagnetic iron oxide particles are
obtained, and [0076] d. Contacting the ferrimagnetic iron oxide
particles with a silicate solution such that silanized
ferrimagnetic iron oxide particles are obtained. [0077] 2. The
method of item 1, wherein contacting the ferrimagnetic iron oxide
particles with a silicate solution comprises the steps of: [0078]
d1. Sonificating the silicate solution comprising the ferrimagnetic
iron oxide particles, [0079] d2. Lowering the pH of the silicate
solution to a value of 6 or below such that silanized ferrimagnetic
iron oxide particles are obtained, [0080] d3. Washing of the
silanized ferrimagnetic particles with water, and [0081] d4.
Washing of the silanized ferrimagnetic particles with isopropanol
such that crosslinking occurs within the silicate layer. [0082] 3.
Method of items 1-2, wherein the iron(II) salt is soluble in the
liquid glycol. [0083] 4. Method of item 3, wherein the iron(II)
salt soluble in the liquid glycol is selected from the group
consisting of FeCl.sub.2, FeSO.sub.4, FeAc.sub.2 and the hydrated
forms thereof. [0084] 5. Method of items 1 to 4, wherein the liquid
glycol is an alkyl glycol and polymerized forms thereof. [0085] 6.
Method of item 5, wherein the alkyl glycol is selected from the
group consisting of ethylene glycol, diethylene glycol, triethylene
glycol, tetraethylene glycol, propylene glycol, dipropylene glycol,
tripropylene glycol, tetraproylene glycol, butylene glycol,
dibutylene glycol, tributylene glycol, tetrabutylene glycol. [0086]
7. Method of item 5, wherein the alkyl glycol is triethylene
glycol. [0087] 8. Method of items 1-7, wherein the pH of the
solution in step b) is raised to a value of 10.5. [0088] 9. Method
of items 1-8, wherein the pH of the solution in step b) is raised
using sodium hydroxide. [0089] 10. Method of items 1-9, wherein the
first temperature has a value from 20 to 120.degree. C. [0090] 11.
Method of item 10, wherein the first temperature has a value of
100.degree. C. [0091] 12. Method of item 11, wherein the first
temperature is maintained for 5 to 60 min. [0092] 13. Method of
items 1-12, wherein the second temperature has a value from 150 to
300.degree. C. [0093] 14. Method of item 13, wherein the second
temperature has a value of 250.degree. C. [0094] 15. Method of item
14, wherein the second temperature is maintained for 20 min to 48
h. [0095] 16. Method of items 1-15, wherein the silicate is
selected from the group consisting of potassium silicate and sodium
silicate. [0096] 17. Method of items 1-16, wherein the silicate is
present in the solution in a concentration from 1 to 30% w/v.
[0097] 18. Method of item 17, wherein the silicate is present in
the solution in a concentration of 5.3% w/v. [0098] 19. Method of
items 2-18, wherein the pH of the solution in step d2) is lowered
to a value of 5. [0099] 20. Method of item 19, wherein the pH of
the solution in step d2) is lowered using an acid selected from the
group consisting of phosphoric acid, sulphuric acid and
hydrochloric acid. [0100] 21. Method of item 20, wherein the pH of
the solution in step d2) is lowered using hydrochloric acid. [0101]
22. Method of items 1-21, wherein step d) is repeated one or more
times. [0102] 23. Monodisperse silanized ferrimagnetic iron oxide
particles for independent generic nucleic acid binding having the
following characteristics: [0103] a. A core, wherein the core
comprises an inner layer comprising Fe.sub.3O.sub.4 and an outer
layer comprising Fe.sub.2O.sub.3, [0104] b. A coating, wherein the
coating comprises silica and silicates from sodium silicate
precipitation, [0105] c. A sedimentation speed in pure water of
less than 60 .mu.m/s, and [0106] d. No significant iron bleeding
occurs on the particles in 1M HCl for at least 60 min. [0107] 24.
The monodisperse silanized ferrimagnetic iron oxide particles
according to item 23, wherein the difference in size of the
monodisperse silanized ferrimagnetic iron oxide particles is in
average smaller than 5%. [0108] 25. The monodisperse silanized
ferrimagnetic iron oxide particles according to item 24, wherein
the size of the particles n is between 20 nm and 600 nm. [0109] 26.
Method for independent generic binding of nucleic acid molecules by
use of the monodisperse silanized ferrimagnetic iron oxide
particles of items 23-25. [0110] 27. Method of item 26, wherein the
nucleic acid molecules are DNA molecules or RNA molecules.
EXAMPLES
[0111] The following examples 1 to 4 are provided to aid the
understanding of the present invention, the true scope of which is
set forth in the appended claims. It is understood that
modifications can be made by a person of ordinary skill in the art
to the procedures set forth below.
Example 1
Cleaning of the Reactor
[0112] The reactor used in the present description is a Buchi
Midiclave Reactor with propeller stirrer and stream breaker from
Hasteloy Steel.
[0113] Immediately after the removal of the ironoxide nanoparticle
suspension, the flow break, agitator shaft and temperature sensor
were washed 3 times with water.
[0114] The reactor was filled with 900 mL of a 0.02 M EDTA and
0.0067 M FeCl.sub.2 solution and was heated to 120.degree. C. and
stirred with 1000 rpm for 15 h. The gasket ring between top panel
and agitator vessel was removed and swiveled in 32% HCl for 30
seconds, than washed for 30 seconds under floating VE-H.sub.2O.
[0115] Temperature sensor, N.sub.2-pipe, flow brake and agitator
shaft are rinsed with VE-H.sub.2O. The stirrer was removed and
disassembled completely. The disassembled parts are shown and
labeled in FIG. 2. The dissassembling was performed by the
following steps: i) Remove wire on the top of the stirring engine,
ii) pull the agitator shaft out, iii) unscrewe the stirring engine
together with the shaft, iv) unscrewe the shaft out of the stirring
engine (use the steel stick), v) remove gasket ring between top
panel and shaft, vi) remove circular spring, vii) remove the upper
sleeve bearing (use the fork), viii) release the binding screw
between inner magnet and the agitator shaft and separate the parts,
ix) strip down the lower sleeve bearing, and x) release the
agitator blade screw and remove the agitator blade.
[0116] The shaft was washed for 30 seconds under floating
VE-H.sub.2O. In case of remaining impurities (brown/dark spots) in
the shaft, the shaft was filled with HCl and subsequently floated
with VE-H.sub.2O. Then, the shaft was screwed back into the
stirring engine. The swing gasket ring between top panel and shaft
was rinsed for 30 seconds in 32% HCl and floated in VE-H.sub.2O for
30 seconds. The circular spring was washed for 30 seconds with
VE-H.sub.2O and bigger particles were removed using forceps.
[0117] The inner magnet, binding screw, agitator shaft, stirring
blade, stirring blade screw, upper and lower sleeve were rinsed in
30 seconds in 32% HCl and washed afterwards under floating
VE-H.sub.2O. The agitator shaft and the stirring blade are rebuilt
with the stirring blade screw. The guide slot was lubrified with
glycerin, put on the lower sleeve bearing and further lubricated on
its outside. The inner magnet and the agitator shaft was assembled
with the binding screw. The upper sleeve bearing was lubricated in
its inside, put it on the inner magnet until it locks and then
lubricated with glycerin on its outside. The flow break and the
flow break pole was removed, rinsed them together with the screw
nut in 32% HCl for 30 seconds, than wash it under floating
VE-H.sub.2O for 30 seconds.
[0118] The outlet N.sub.2-sleeve was removed. The sleeve clamp was
loosened and the metal binding piece was pulled out. The binding
piece was rinsed for 30 seconds in 32% HCl, then washed under
floating VE-H.sub.2O for 30 seconds. Subsequently, the binding
piece was inserted in the sleeve and fixed with the sleeve
clamp.
[0119] As a next step, manometer, temperature sensor, N.sub.2-pipe,
needle valve and burst protection was removed. The respective parts
are shown and labeled in FIG. 3. Flush the manometer with
VE-H.sub.2O. The burst protection, needle valve and the
N.sub.2-pipe was cleaned with a cleaning cloth soaked in 32% HCl,
followed by flushing it with a disposable pipette until the out
coming HCl had no more visible colouring and washing it inside and
outside under floating VE-H.sub.2O for 30 seconds.
[0120] The temperature sensor was cleaned with a cleaning cloth
soaked in 32% HCl followed by washing it for 30 seconds with
VE-H.sub.2O.
[0121] The three screws holding the top panel were solved and
removed. Subsequently, the three allen keys which fix the core of
the top panel were solved. See FIG. 4 showing the corresponding
parts.
[0122] The core of the top panel (especially the seven screw holes)
was cleaned with a disposable pipette with 32% HCl. The cleaning
step was performed until the HCl did not show any visible yellow
coloring. The core of the top panel was then washed under floating
VE-H.sub.2O for 30 seconds.
[0123] The two pressure sleeves at the agitator vessel were
removed. The agitator vessel was washed for 90 seconds with 32%
HCl. Subsequently, the agitator vessel was filled with 100 ml of
32% HCl and cleaned with a chemical resistant brush for 60 seconds.
The HCl from the agitator vessel was discarded into a beaker. The
last cleaning step was repeated twice. The agitator vessel was
washed again for 90 seconds with 32% HCl. Finally, the agitator
vessel was reassembled with the two pressure sleeves.
[0124] The core of the top panel was reassembled with the flow
break. Then the manometer, temperature sensor, N.sub.2-pipe, needle
valve and burst protection was mounted back into the top panel. The
gasket ring was placed between the shaft and the top panel. The
stirring engine and the agitator shaft was inserted. The gasket
ring was placed between top panel and agitator vessel.
[0125] The success of the cleaning procedure was controlled by
filling the reassembled reactor with 1L VE-H.sub.2O. The reactor
was heated to 150.degree. C. for 60 minutes with 500 rpm The step
was repeated three times. After the third time of boiling, the
VE-H.sub.2O should have been clear, otherwise the cleaning
procedure was repeated.
Example 2
Manufacturing of the Ferrimagnetic Iron Oxide Particles
[0126] Reaction equation:
3 FeCl.sub.2+6 NaOH->Fe.sub.3O.sub.4+6 NaCl+H.sub.2+2
H.sub.2O
[0127] 400 mL (4.21 mol) of triethylenglycol were put in a
glas-reactor. While the reactor was heated to 100.degree. C.,
stirring with the magnetic stirrer and introducing N.sub.2
(pressure 0.4 bar at the outlet valve) into the triethylenglycol
for 30 minutes was performed. Subsequently, 12.31 g FeCl.sub.2*4
H.sub.2O (60.8 mmol) and 12 mL NaOH (10 M) (120 mmol) were added to
the triethylenglycol at 100.degree. C. and still introducing
N.sub.2 while strengthen stirring (if necessary rise stirring
speed) was continued for 10 minutes.
[0128] Transferring the glas-reactor content into a reactor which
was preheated to 80.degree. C. Stirring was performed for 10
minutes without further heating and introducing N.sub.2. During
continued stirring, the reactor was heated according to the
protocol as depicted in FIG. 1. The temperature was increased from
the preheated temperature of 80.degree. C. to a value of
250.degree. C. within a time period of 20 min. The value of
250.degree. C. was maintained for a time period of 80 min.
Subsequently, the second temperature was decreased to a value of
30.degree. C. within a time period of 35 min.
[0129] After the reactor temperature reached 30.degree. C. the
pressure was released and the suspension was put into a beaker. The
suspension was dialyzed over night in a 36/32 inch sleeve in
Milli-Q-H.sub.2O. The sleeve was soaked and washed for 10 minutes
with Milli-Q-H.sub.2O before filling it with particels. After
dialyzing over night, the particels were filled into a Schott glas
bottle. After sedimentation of the particles and removing the
supernatant to a total volume of 50 mL, the suspension was
transferred into a falkon tube.
Example 3
Silanizing the Ferrimagnetic Iron Oxide Particles
[0130] 300mL of a 5.3% w/v silane solution were provided in a
special design glas reactor 8 mL of the particle solution (0.5 g
particle) as manufactured according to Example 2 were added. A
schematic drawing of the special design glas reactor is presented
in FIG. 5 (Ar=Argon, M=Motor, US=Ultrasonic probe). Sonification of
the solution was performed using an ultrasonic probe every 50
seconds 5 times with 80% of the maximal amplitude energy intake for
200 ms. Subsequently, 100 mL of 1 M HCl was added followed by
stirring at 300 rpm and continued use of the ultrasonic probe over
night (with the same settings as mentioned above). Then, 10 mL of 1
M HCl was added and the solution was stirred for two more hours and
an argon source drain was attached to the reactor to avoid oxygen
in the atmosphere covering the solution. After sedimentation, the
particles were washed with 400 mL VE-H.sub.2O. After a further
sedimentation of the particles, the supernatant was discarded.
Subsequently, 300 mL silane solution was added to the particles.
Sonification of the solution was performed every 50 seconds 5 times
with 80% of the maximal amplitude energy intake for 200 ms.
Subsequently, 100 mL of 1 M HCl was added followed by stirring at
300 rpm and continued use of the ultrasonic probe over night (same
settings as mentioned above). Then, 10 mL of 1 M HCl was added and
the solution was stirred for two more hours. After sedimentation,
the particles were washed with 400 mL VE-H.sub.2O. After a further
sedimentation of the particles, the supernatant was discarded. The
washing procedure was repeated 5 more times.
Example 4
Use of the Monodisperse Silanized Ferrimagnetic Iron Oxide
Particles for Independent Generic Nucleic Acid Binding
[0131] Monodisperse silanized ferromagnetic iron oxide particles
produced according to the method described herein as exemplified in
the Examples 1 to 3 were used for binding target nucleic acids from
two different clinically relevant pathogenic organisms.
[0132] The particles exhibited different degrees of silanization,
meaning that different relative amounts of coating were present on
the particle surfaces. More specifically, the different percentages
indicated in the tables below represent the mass of coating in
relation to the mass of the respective iron oxide core. Hence, a
suffix of "80%" denotes a population of particles wherein the
coating mass is 80% of the iron oxide core mass.
[0133] Further, one set of particles was coated with two layers of
silane, having been subjected to two consecutive rounds of
silanization as described herein.
[0134] As a reference, magnetic glass particles from the
commercially available MagNA Pure 96 DNA and Viral NA Large Volume
Kit (Roche Diagnostics, Catalog No. 06374891001) were used in
parallel experiments. Also, the reagents from the above-mentioned
kit were used in a nucleic acid isolation procedure following the
instructions of the user manual. The person skilled in the art is
able to readily apply other procedures for using the particles
described herein for binding nucleic acids. It is to be noted that
for each experiment only 1 mg of the particles described herein
were used, while the particles provided with the above-mentioned
kit had to be used in portions of 12 mg for each experiment,
according to the user manual.
[0135] After isolating the respective nucleic acids, they were
subjected to realtime PCR on a LightCycler.RTM. 480 Analyzer (Roche
Diagnostics) exploiting the commercially available kits listed in
the following.
[0136] Isolation of Parvo B19 Virus (DNA)
[0137] Equipment:
TABLE-US-00001 Name Catalog. No. Company Parvo B 19 Quantification
Kit 0324680 809 Roche Diagnostics
[0138] PCR results:
TABLE-US-00002 Coating Coating Coating Twice Twice to core to core
to core 1 Kit Kit silanized silanized 00% 80% 20% particles
particles Particles run1 run2 [W/W] [W/W] [W/W] run1 run2 cp-mean
25.37 25.64 26.05 27.86 25.56 26.54 25.28 cp-min 25.22 25.59 25.97
27.71 24.99 26.54 25.28 cp-max 25.52 25.69 26.10 28.02 25.84 26.54
25.28 .DELTA. cp max - 0.3 0.1 0.1 0.3 0.9 0.0 0.0 cp min
Replicates 2 2 4 3 5 1 1
[0139] Isolation of Influenza Virus (RNA)
[0140] Equipment:
TABLE-US-00003 Name Catalog No. Company Real Time ready RNA
VirusMaster 05 619 416 001 Roche Diagnostics Real Time ready
Influenza A 05 640 393 001 Roche Diagnostics H1N1 Detection Set
[0141] PCR results:
TABLE-US-00004 Coating Coating Coating Twice Twice to core to core
to core Kit Kit silanized silanized 100% 80% 20% particles
particles Particles run1 run2 [W/W] [W/W] [W/W] run1 run2 cp-mean
29.12 29.14 32.48 34.73 31.31 29.90 29.97 cp-min 29.00 29.04 32.29
34.52 31.21 29.84 29.72 cp-max 29.23 29.24 32.65 34.90 31.44 29.96
30.22 .DELTA. cp max - 0.2 0.2 0.4 0.4 0.2 0.1 0.5 cp min
Replicates 2 2 4 4 4 2 2
[0142] The results clearly show that the particles produced by the
method described herein are at least equivalent to the commercially
available reference particles in their capacity to bind and thereby
isolate target nucleic acids, comprising both DNA and RNA. Notably,
the respective Cp (Crossing point) values which, in the current
experimental setting, provide a measure for the nucleic acid yield
following the binding/isolation procedure, are overall even
improved. This is particularly evident for the twice-silanized
particles produced by the method described herein, even though in
all instances the mass of particles used was only a small fraction
(1:12) of the mass of prior art particles.
[0143] In summary, it has been shown that the monodisperse
silanized ferrimagnetic iron oxide particles of the present
invention can be used for independent generic nucleic acid binding
with properties that are as well or even better than particles used
in the prior art, while displaying further advantageous properties
with regard to their efficiency and their production procedure.
* * * * *